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First published online 18 February 2009
doi: 10.1242/dev.029926

Development 136, 1093-1104 (2009)
Published by The Company of Biologists 2009
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BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation

Kelsey N. Retting1,2, Buer Song1, Byeong S. Yoon1,3 and Karen M. Lyons1,2,3,*

1 Orthopaedic Hospital/UCLA Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
2 Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
3 Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA.


Figure 1
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  		Fig. 1. Cartilage-specific excision of mouse Smad1 and Smad5. Smad1 and Smad5 are excised in cartilage of Smad1/5CKO mutants. (A,B) Immunofluorescence analysis of C-terminal Smad phosphorylation (pSmad1/5) in wild-type (WT) (A) and mutant (B) cartilage, counterstained with DAPI. Arrows demarcate the border of the perichondrium. In the mutant, blood vessels autofluoresce red and delineate the location of the perichondrium. (C) Western blot analysis of microdissected WT and mutant growth plate lysates for total and phosphorylated forms of Smad1 and Smad5 (C-terminus, pSmad15C; linker region, pSmad1L). (D,E) Immunofluorescence of pSmad1/5 in cultured WT (D) and mutant (E) primary sternal chondrocytes.

 

Figure 2
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  		Fig. 2. Overlapping functions for Smad1 and Smad5 in cartilage. All cleared skeletal preps are of P0 mice. (Above) Mice lacking Smad1, Smad5 or Smad8 are viable and indistinguishable from WT littermates. (Below) Mice lacking Smad1 and Smad8 are viable and indistinguishable from WT littermates. Mice lacking Smad1 and Smad5 (Smad1CKO;Smad5CKO) and Smad1/5/8 triple mutants (Smad1CKO;Smad5CKO;Smad8-/-) exhibit severe chondrodysplasia.

 

Figure 3
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  		Fig. 3. Chondrodysplasia in Smad1/5 double mutants and Smad1/5/8 triple mutants. (A,B) Alcian Blue-stained sections through E16.5 vertebrae of WT and Smad1/5CKO double-mutant littermate mice. (C,D) MicroCT analysis of P0 WT and Smad1/5CKO littermates. Sternebrae are present, but malformed (arrow). Cortical bone is evident in appendicular elements of the mutant (arrowhead). (E-G) Alcian Blue-stained sections through E16.5 proximal tibiae. Sections through tibial condensations were smaller in triple mutants than in double mutants, but were otherwise indistinguishable.

 

Figure 4
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  		Fig. 4. Defective limb development in Smad1/5CKO mutants. (A,B) WT (A) and mutant (B) Alcian Blue-stained E12.5 mouse limb cartilage. Brackets identify perichondrium. (C,D) WT (C) and mutant (D) proximal tibial growth plate at E14.5. (E,F) Immunofluorescence of type I collagen (red) and type II collagen (green) counterstained with DAPI (blue) in E14.5 WT (E) and mutant (F). (G-J) Alcian Blue and Von Kossa staining at E16.5 in WT (G,I) and mutant (H,J) proximal tibia, respectively. The arrows mark the bony collar in I,J. (K,L) Immunofluorescence of type I (red) and type II (green) collagen at E17.5 in WT (K) and mutant (L) proximal tibia. The inset in L is an enlargement of the boxed region showing type II collagen-producing cells embedded in the mutant type I collagen expression domain. (M,N) Alcian Blue staining of P0 WT (M) and mutant (N) knee joint. The arrow indicates ectopic cartilage formation in mutants; the asterisk demarcates a small marrow cavity.

 

Figure 5
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  		Fig. 5. Growth plate disorganization and impaired chondrocyte survival in Smad1/5CKO mutants. (A,B) Autofluorescence (green) and DAPI (blue) staining in E17.5 WT (A) and mutant (B) mouse proximal tibial growth plates. (C,D) Safranin O staining of E17.5 WT (C) and mutant (D) tibial growth plates. Double-headed arrows demarcate the borders of the perichondrium. (E,F) Pcna immunofluorescence of WT (E) and mutant (F) proximal tibiae. Arrows indicate staining limited to perichondrium and lateral edges of the mutant cartilage element. (G,H) TUNEL staining in E17.5 WT (G) and mutant (H) proximal tibiae. Images in C, E and G are adjacent sections; images in D, F and H are adjacent sections.

 

Figure 6
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  		Fig. 6. Impaired matrix production and chondrocyte differentiation in Smad1/5CKO mutants. (A-H) Adjacent sections of WT (A,C,E,G) and mutant (B,D,F,H) E17.5 mouse cartilage. (A,B) Safranin O staining. (C,D) Immunofluorescence for aggrecan. (E,F) Immunofluorescence for type II collagen in growth plates. Arrow indicates restricted type II collagen production in the mutant. (G,H) Immunofluorescence for type X collagen in growth plates. (I,J) RT-PCR analysis of gene expression using RNA isolated from microdissected WT and mutant cartilage.

 

Figure 7
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  		Fig. 7. The BMP canonical Smad pathway is required for the Ihh/PTHrP signaling loop. (A) RT-PCR analysis of WT mouse primary chondrocytes treated (+), or otherwise (-), with BMP2 for 2 hours. (B) RT-PCR analysis of RNA isolated from microdissected E18.5 WT and mutant growth plate cartilage. (C-F) In situ hybridization analysis of Ihh expression in E16.5 WT (C,D) and Smad1/5CKO (E,F) proximal tibiae. (G-J) In situ hybridization analysis of Pthrp expression in WT (G,H) and Smad1/5CKO (I,J) proximal tibiae. Arrowhead in H demarcates the perichondrium/periosteum. (K,L) Immunostaining for PPR in WT (K) and Smad1/5CKO (L) proximal tibiae. (M-P) In situ hybridization for Ptch1 expression, illustrating strong expression in WT proliferating chondrocytes, and a weaker signal in the mutant growth plate (P, arrowhead). Arrow in P highlights higher levels of Ptch1 expression in mutant osteoblasts and periosteum.

 

Figure 8
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  		Fig. 8. Imbalance of BMP and FGF signaling in mutant cartilage. (A,B) Immunofluorescence staining for Fgfr1 in WT (A) and Smad1/5CKO;Smad8+/- mutant (B) E17.5 mouse proximal tibiae. (C,D) Total Stat1 immunofluorescence in WT (C) and mutant (D) E16.5 proximal tibiae. (E,F) High-magnification images of the boxed regions from C and D showing subcellular localization of Stat1 (arrows) in WT (E) and mutant (F) chondrocytes.

 

Figure 9
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  		Fig. 9. FGF inhibits BMP activity in primary chondrocytes and RCS cells. (A) BMP2 induction of the mouse 1.8 kb Msx2 promoter is inhibited by FGF2 in a dose-dependent manner in RCS cells. (B) An Erk1/2 MAPK inhibitor (PD98059) abrogates the inhibitory effects of FGF2 on BMP2 activity in RCS cells. (C) The phosphorylation (P) sites in the Smad constructs Smad1WT and Smad1LM. (D) BMP2 induction of the 1.8 kb Msx2-luc construct is enhanced by transfection of Smad1WT or Smad1LM. (E) The Ihh promoter is inhibited by FGF, and mutation of the Smad1 linker region can prevent these inhibitory effects. Each transfection experiment was repeated at least three times and a representative experiment is shown in each panel. The data represent an average from three wells with the indicated s.d. Brackets with an asterisk indicate significant differences between columns (Student's t-test; P≤0.05). All other asterisks indicate significant differences from the no treatment control. NS, not significant.

 

Figure 10
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  		Fig. 10. C-terminal and linker phosphorylation of BMP receptor Smads in chondrocytes. (A-H) Immunostaining of radius and ulna of E16.5 cultured mouse limbs treated with FGF18 or the FGF receptor antagonist SU5402. For each experiment, the contralateral limb served as an untreated control. (A,B) Immunofluorescence images of phosphorylated (p) Smad1/5 expression in untreated control (A) and contralateral limb treated with FGF18 (B). (C,D) Immunofluorescence images of pSmad1/5 expression in untreated control (C) and contralateral limb treated with SU5402 (D). (E,F) Immunofluorescence images of pSmad1L expression in untreated control (E) and contralateral limb treated with FGF18 (F). (G,H) Immunofluorescence images of pSmad1L expression in untreated control (G) and contralateral limb (H). (I) Western blot showing that although FGF18 induces pErk1/2, it does not induce Smad1 linker phosphorylation. Rather, Smad1 linker phosphorylation is induced by BMP2. (J) Immunofluorescence images of pSmad1/5 and pSmad1L localization in primary chondrocyte cultures. Both forms of Smad1 are primarily nuclear.

 

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© The Company of Biologists Ltd 2009